Effects of experimental procedures and conditions on bioassay sensitivity of alfalfa autotoxicity

Sampling procedures of the donor material, conditions of the assay, and sel ection of test organism influence the sensitivity of a bioassay. Autotoxici ty affects success of alfalfa (Medicago sative L.) reseeded after old alfal fa. Root length is a more sensitive parameter to autotoxin than germination or hypocotyl length. Our specific objective was to improve the sensitivity of an alfalfa seedling bioassay for a better understanding of the autotoxi city mechanism. Using a petri-dish assay with imbibed seed, we found extrac t of alfalfa leaves was more autotoxic than either that of stems or seed. E xtracts from oven-dried samples were stronger than those from freeze-dried samples. Sterilization through membrane filtering or autoclaving did not al ter autotoxicity bioassay. Root length at 25 or 50 seed/plate was more sens itive to the extracts than that at 100 or 200 seed/plate. Roots from older seedlings were more tolerant to the extracts than did those from seed or yo unger seedling and had more branch roots to escape the autotoxin(s). These results suggest that extracts from leaf samples that oven-dried and sterili zed by filtering or autoclaving can improve the consistency and precision o f bioassay. The bioassay was suitable with 25 to 50 seed per petri dish to minimize interaction on interference (e.g., competition and autotoxicity fr om testing seed itself). The bioassay can be improved by using very young r oots from imbibed seed which are more sensitive to the autotoxin.